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Myosin is the principal protein of the myofilaments in muscle. Along with actin, it is involved in muscle contraction. Myosin, from several species, has been purified and characterized. Recent studies on highly purified myosin indicate that it has a molecular weight of about 500,000. Purified myosin preparations have the capacity to hydrolyze the terminal phosphate group from adenosine triphosphate (ATP). The reaction can be illustrated as follows:

ATP ------> ADP + Pi
The ATPase activity of myosin is stimulated by Ca2+ and it has two pH optima, one at pH 6.0 and the other at pH 9.5. The reaction is also influenced by KCl concentration. In addition to ATP, myosin can also hydrolyze the terminal phosphate groups from cytidine triphosphate (CTP), guanosine triphosphate (GTP) and inosine triphosphate (ITP).

The amount of myosin that will liberate one micromole of inorganic phosphorous from ATP per minute at 25°C, pH 9.0 in the presence of calcium ions.

The catalytic activity of myosin is determined by estimating spectrophotometrically the inorganic phosphate liberated by the enzyme from ATP in the presence of calcium ions (Perry, S.V. in Methods in Enzymology, Colowick, S.P. and Kaplan, N.O. eds.; Vol II, 582, 1955, Academic Press, New York).

  1. 0.2 M Glycine-NaOH buffer, pH 9.0.
  2. 0.1 M Calcium chloride in distilled water.
  3. 0.05 M ATP, sodium salt, pH 6.8.
  4. Myosin (enzyme) solution. Make appropriate dilutions using 0.5 M KCl.
  5. 15% Trichloroacetic acid (TCA).

  1. In the required number of test tubes pipette the following reagents in the amounts indicated:
    Glycine-NaOH buffer 1.3 ml
    Calcium chloride 0.2 ml
    ATP 0.3 ml
  2. Incubate test tubes at 25°C for 5-10 minutes.
  3. Initiate reaction by addition of 0.2 ml myosin solution to each test tube (to the blank tube add 0.2 ml 0.5 M KCl solution). Place in water bath-shaker at 25°C for 5 minutes.
  4. Terminate reaction by rapid addition of 1 ml 15% TCA to each tube.
  5. Centrifuge to remove precipitate.
  6. Use 1 ml supernatant for the estimation of inorganic phosphate released (Fiske, C.H. and Subba Row Y., J. Biol. Chem., 66, 375, 1925).

Calculate units of activity for myosin by using a phosphate standard curve.

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